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Rapid sensitive detection method of GGC repetitive sequence of NOTCH2NLC gene and application of rapid sensitive detection method

A repetitive sequence and gene technology, applied in the medical field, can solve the problems of misjudgment of test results and high proportion of CG in amplified fragments, and achieve the effects of rapid detection, reduced detection cost and sensitive detection

Active Publication Date: 2021-09-03
THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Based on PCR amplification of the NOTCH2NLC gene, the resolution of the amplified fragments detected by capillary electrophoresis is higher than that of traditional agarose gel electrophoresis, but because the CG ratio of the amplified fragments is too high, the difficulty increases with the number of repetitions. The design of primers, because the PCR reaction system is not adjusted to the optimal conditions, may lead to misjudgment of the test results, and the PCR time is more than 10 hours[2]

Method used

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  • Rapid sensitive detection method of GGC repetitive sequence of NOTCH2NLC gene and application of rapid sensitive detection method
  • Rapid sensitive detection method of GGC repetitive sequence of NOTCH2NLC gene and application of rapid sensitive detection method
  • Rapid sensitive detection method of GGC repetitive sequence of NOTCH2NLC gene and application of rapid sensitive detection method

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Embodiment 1

[0030] This embodiment discloses a method for rapidly detecting the GGC repeat sequence of the NOTCH2NLC gene, which specifically includes the following steps:

[0031] 1. Obtain the genomic DNA of the sample to be tested;

[0032] 2. Using the amplification reaction system to amplify the NOTCH2NLC gene;

[0033] 3. Detect the repetitive sequence GGC of the NOTCH2NLC gene in the obtained amplification product.

[0034] Wherein the amplification reaction system includes a primer composition, which is composed of an upstream primer, a downstream primer and a third primer of a non-human gene sequence, such as figure 1 As shown, the specific sequence is shown in Table 1.

[0035] Table 1

[0036]

[0037] The amplification reaction system is shown in Table 2 below.

[0038] Table 2

[0039]

[0040] Citing the document 2 program, that is, the amplification program is:

[0041] React at 98°C for 10min; then react at 98°C for 30sec, 66°C for 1min (each cycle reduces 0.5°...

Embodiment 2

[0044] The only difference between the method of this embodiment and embodiment 1 is that the amplification procedure is:

[0045] React at 98°C for 10min; then react at 98°C for 30sec, 66°C for 1min (each cycle reduces 0.5°C), and 68°C for 1min, a total of 20 cycles; then react at 98°C for 30sec, 56°C for 30sec, and 68°C React for 1 min, a total of 15 cycles; last 10 minutes at 68°C. The adjustment of all cycle steps is 0.5°C / sec, which is the new program.

[0046] It was found that the new procedure of the innovative primers of the present invention (as shown in Table 1 above) well solved the PCR amplification of the abnormal repeat of 5'UTR GGC in the NOTCH2NLC gene, as follows Figure 4 As shown, it was found that the procedure of 1 hour and 40 minutes can complete the PCR amplification of the 5'UTR GGC abnormal repeat in the NOTCH2NLC gene.

Embodiment 3

[0048] This example studies the sensitivity of the innovative primers to the 5'UTR GGC abnormal repeat amplification in the positive control NOTCH2NLC gene with different templates, and the results are as follows image 3 shown. Among them, the DNA samples (5ng-300ng) of the positive control patients were amplified by PCR with the literature primers and the innovative primers, and the capillary electrophoresis results were compared, and it was found that the innovative primers were more sensitive, and positive signals could be detected at 5ng.

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Abstract

The invention relates to the field of medicine, in particular to a rapid sensitive detection method of the GGC repetitive sequence of a NOTCH2NLC gene and an application of the rapid sensitive detection method. Firstly, the invention discloses a primer composition for detecting the GGC repetitive sequence of the NOTCH2NLC gene, and the primer composition comprises an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 2 and a third primer with a nucleotide sequence as shown in SEQ ID NO: 3. The invention discloses a novel primer composition for detecting a sample with a high GGC repetition number, optimizes a PCR detection program, is more sensitive to the detection of the sample with the high GGC repetition number, can accurately and quantitatively complete the detection more quickly, shortens the PCR time from 10 hours to less than 2 hours, effectively reduces the workload of operators and reduces the detection cost.

Description

technical field [0001] The invention relates to the field of medicine, in particular to a rapid and sensitive detection method for GGC repeat sequence of NOTCH2NLC gene and application thereof. Background technique [0002] Neuronal intranuclear inclusion disease (NIID) is a very rare disease. Early case reports were common in European and American populations, and it was more common in children and adolescents. Later, it was found that Asian adults were more common. The clinical manifestations of NIID are complex and diverse, including dementia, ataxia, abnormal behavior, tremor, and epilepsy. In 2019, it was discovered that the 5'UTR GGC abnormal repeat expansion in the NOTCH2NLC gene is related to NIID, and subsequently it was found to be related to various neurodegenerative diseases such as Alzheimer's disease and frontotemporal dementia [1] . [0003] Currently, the diagnosis of NIID mainly includes classic P62 and UB-positive eosinophilic nuclear inclusions and NOT...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6883C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 孙淼王彬徐兴顺鲁礼魁
Owner THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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